Increased memory differentiation is associated with decreased polyfunctionality for HIV but not for cytomegalovirus-specific CD8+ T cells

Abstract

The generation of polyfunctional CD8(+) T cells, in response to vaccination or natural infection, has been associated with improved protective immunity. However, it is unclear whether the maintenance of polyfunctionality is related to particular cellular phenotypic characteristics. To determine whether the cytokine expression profile is linked to the memory differentiation stage, we analyzed the degree of polyfunctionality of HIV-specific CD8(+) T cells within different memory subpopulations in 20 antiretroviral therapy-naive HIV-1-infected individuals at ∼34 wk postinfection. These profiles were compared with CMV-specific CD8(+) T cell responses in HIV-uninfected control subjects and in individuals chronically infected with HIV. Our results showed that the polyfunctional abilities of HIV-specific CD8(+) T cells differed according to their memory phenotype. Early-differentiated cells (CD45RO(+)CD27(+)) exhibited a higher proportion of cells positive for three or four functions (p < 0.001), and a lower proportion of monofunctional cells (p < 0.001) compared with terminally differentiated (TD; CD45RO(-)CD27(-)) HIV-specific CD8(+) T cells. The majority of TD HIV-specific CD8(+) T cells were monofunctional (median 69% [interquartile range: 57-83]), producing predominantly CD107a or MIP1β. Moreover, proportions of HIV-specific monofunctional CD8(+) T cells positively associated with proportions of TD HIV-specific CD8(+) T cells (p = 0.019, r = 0.54). In contrast, CMV-specific CD8(+) T cell polyfunctional capacities were similar across all memory subpopulations, with terminally and early-differentiated cells endowed with comparable polyfunctionality. Overall, these data show that the polyfunctional abilities of HIV-specific CD8(+) T cells are influenced by the stage of memory differentiation, which is not the case for CMV-specific responses.

Figure 1. Polyfunctional and memory maturation profiles of HIV-specific CD8+ T cells

(A) Magnitude of total cytokine production (CD107a, MIP1β, IFNγ or TNFα) in CD8+ T cells in response to HIV Gag, Env, Nef and Pol peptide pools. Results are expressed as % of total memory CD8+ T cells. Numbers correspond to the number of positive responses/number of tested individuals. (B) Polyfunctional profiles of HIV-specific CD8+ T cells. All possible combinations of four functions (CD107a, MIP1β, IFNγ and TNFα) produced by Gag, Env, Nef and Pol-specific CD8+ T cells are shown on the x-axis. Box and whiskers indicate the median percentage and range of the total response contributed by CD8+ T cells. Functional profiles are grouped and color-coded according to number of functions and summarized in the pie charts. Each slice of the pie corresponds to the median production of 4 (red), 3 (orange), 2 (green) or 1 (blue) function. (C) Representative dot plot of the memory maturation profile of total CD8+ T cells (density) and Gag-specific total cytokine+ CD8+ T cells (red dots) from one participant. ED: Early-differentiated memory cells (CD45RO+CD27+), LD: Late-differentiated memory cells (CD45RO+CD27−), Inter: Intermediate memory cells (CD45RO−CD27dim) and TD: Terminally-differentiated memory cells (CD45RO−CD27−). (D) Distribution of HIV-specific CD8+ T cells amongst memory subsets, showing 43 HIV-specific responses measured in 20 participants. Closed circles (●) correspond to Gag responses, open circles (○) represent Env responses, crosses (X) correspond to Nef responses and black squares (■) represent Pol responses. Horizontal lines indicate median values.

Figure 2. Polyfunctional profile of HIV-specific CD8+ T cells in defined memory subsets

(A) Representative density and overlay dot plots of memory maturation in total CD8+ T cells (density) and Gag-specific CD8+ T cells endowed with 4 (red), 3 (orange), 2 (green) or 1 (blue) functions for two study individuals with a high and low Gag response. (B) Proportions of HIV-specific CD8+ T cells, exhibiting 4, 3, 2 or 1 functions, across distinct memory subsets. Responses to Gag, Env, Nef and Pol responses have been pooled (n=43 in 20 individuals). Results are shown as box and whisker (10–90 percentile) plots, with outliers depicted with black dots. (C) Representative example of memory profile of total Gag-YL9-specific CD8+ T cells (producing CD107a, MIP1β, IFNγ or TNFα). The pie chart inlaid within the dot plot corresponds to the polyfunctional profile of total Gag-YL9-specific CD8+ T cells. Adjacent pies represent the degree of polyfunctionality in Gag-YL9-specific CD8+ T cells according to their maturation phenotype. (D) Proportion of HIV-specific CD8+ T cells expressing 4, 3, 2 and 1 functions across distinct memory subsets (n=4 responses, in 3 individuals). Each symbol corresponds to an autologous peptide response. ●: Gag (YL9)-specific response in CAP210; ○ : Vif (WI9)-response in CAP210;X: Env (DV9)-specific response in CAP228 and ■: Nef (KF9)-specific response in CAP239. The median and interquartile ranges are shown for each group. The p-values were calculated using one-way ANOVA non-parametric Kruskal-Wallis test; ***: p<0.001, **: p<0.01, *: p<0.05.

Figure 3. Cytokine combinations produced by HIV-specific CD8+ T cells in defined memory subsets

(A) Proportion of HIV-specific CD8+ T cells producing eight distinct combinations of four functions, shown on the x-axis, across distinct memory subsets. Gag, Env, Nef and Pol responses have been pooled (n=43 in 20 individuals). Results are shown as box and whisker (10–90 percentile) plots, and outliers are depicted with black dots. (B) Mean production of different cytokine combinations in HIV-specific ED, Inter, LD and TD subsets. The p-values depicted on the graph correspond to the comparison between ED and TD subsets, and were calculated using a one-way ANOVA non-parametric Kruskal-Wallis test; ***: p<0.001, *: p<0.05.

Figure 4. Polyfunctional and memory maturation profiles of CMV-specific CD8+ T cell responses

(A) Polyfunctional profiles of CMV-specific CD8+ T cells. All possible combinations of four functions (CD107a, MIP1β, IFNγ and TNFα) produced by HIV-infected (n=6) and HIV-uninfected (n=6) individuals are shown on the x-axis. Box and whiskers indicates the median percentage and interquartile range of the total response contributed by CD8+ T cells. Functional profiles are grouped and color-coded according to number of functions and summarized in the pie charts. Each slice of the pie corresponds to the median production of 4 (red), 3 (orange), 2 (green) or 1 (blue) function. (B) Representative dot plots of the memory maturation profile of total CD8+ T cells (density) and CMV-specific total cytokine+ CD8+ T cells (red dots) in one HIV-infected and one HIV-uninfected individual. (C) Comparison of memory maturation profiles of CMV− and HIV-specific CD8+ T cells. Closed circles (●) correspond to HIV-infected individuals (n=6) and open circles (○) represent HIV-uninfected individuals (n=6). Horizontal lines indicate median values. The p-values were calculated using a one-way ANOVA non-parametric Kruskal-Wallis test; ***: p<0.001, **: p<0.01, *: p<0.05.

Figure 5. Polyfunctional profiles of CMV-specific CD8+ T cells in defined memory subsets

Proportion of CMV-specific CD8+ T cells exhibiting 4, 3, 2 or 1 functions across the different memory subsets. HIV-infected and uninfected individuals have been pooled (n=12). Results are shown as box and whisker (10–90 percentile) plots, and outliers are depicted with black dots. The p-values were calculated using a one-way ANOVA non-parametric Kruskal-Wallis test; *: p<0.05.

Figure 6. Comparison of the polyfunctional profiles of HIV− and CMV-specific CD8+ T cells in defined memory subsets

(A) Proportion of HIV-specific (n=43 responses in 20 individuals) and CMV-specific (n=12) CD8+ T cells producing 4, 3, 2 and 1 function across different memory subsets (ED, Inter, LD and TD). Results are shown as box and whisker (10–90 percentile) plots, with outliers depicted with black dots. Statistical comparisons where determined by a Mann-Whitney non-parametric t-test. (B) Associations between the proportions of antigen-specific CD8+ T cells endowed with 1 or 3 functions (% of total antigen-specific CD8+ T cells) and the proportions of TD antigen-specific CD8+ T cells (% of total antigen-specific CD8+ T cells). HIV-specific responses are shown on the left (n=20 individuals) and CMV-specific responses (n=12) on the right. Statistical associations were performed by a two-tailed non-parametric Spearman rank correlation.